Klaus Hantke

Iron and metal regulation in bacteria.

In Escherichia coli, the iron regulator Fur is regulated by two oxidative-stress response regulators. The generation of dangerous radicals by oxygen and iron is the basis for this dual regulation, which is also found in eukaryotes. The binding of iron-regulated transcripts to apo-aconitase and results of mRNA half-life studies indicate that there is post-transcriptional iron regulation in bacteria, as in eukaryotes. Fur contains two metal-binding sites, one for Zn2+ and one for Fe2+. Zinc uptake systems are regulated by members of the Fur protein family, and zinc is a cofactor. DtxR and related proteins constitute another family of iron regulators, first found in Gram-positive organisms with a high GC content. In organisms with Fur-dependent iron regulation, members of the DtxR family regulate manganese transport.

Regulation of ferric iron transport in Escherichia coli K12: Isolation of a constitutive mutant

SummaryThe lac genes were inserted with phage Mu(Ap, lac) into the fhuA, fepA, cir and tonB genes which specify components of iron uptake systems. The expression of lac in all these operon fusions was controlled by the availability of iron to the cells, thereby facilitating a quick and simple measurement of the expression of the genes listed above. In an iron rich medium under anaerobic conditions all systems were strongly repressed. fhuA was depressed at higher iron concentration than was fepA or cir, and tonB was repressed only under anaerobic conditions and could be induced by iron limitation.Mutants constitutive for the expression of β-galactosidase were selected in a fhuA-lac fusion strain. The outer membrane proteins Cir, FhuA, FecA, 76K and 83K were made constitutively in such mutant strains. Therefore, they were termed fur mutants. In these fur mutant strains, the synthesis of a 19K protein was reduced. Furthermore, it was found that transport of ferric enterochelin and ferrichrome was also constitutive in the fur mutant cells, and that ferric citrate uptake could be induced by only 10 μM citrate in the growth medium in contrast to wild-type cells in which at least 100 μM citrate was necessary. The fepA gene was concluded to be under an additional control, because it was not fully derepressed by the fur mutation.

Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN

Members of a family of catecholate siderophores, called salmochelins, were isolated by reversed-phase HPLC from Salmonella enterica serotype Typhimurium and structurally characterized by Fourier transform ion cyclotron resonance–MS/MS and GC–MS. The tentative structure of salmochelin 1 contained two 2,3- dihydroxybenzoylserine moieties bridged by a glucose residue, bound to the serine hydroxyl group of one moiety and the carboxylate of the second moiety. Salmochelin 2 contained in addition a second glucose residue linked to a third 2,3-dihydroxybenzoylserine moiety. Salmochelins were not produced by an iroBC mutant, which indicated that the IroB protein might be responsible for the glucosyl transfer predicted by sequence similarities to known glycosyltransferases. Uptake experiments with radiolabeled 55Fe-salmochelin and growth promotion tests with salmochelins showed that the IroN outer membrane receptor, encoded in the iroA locus of S. enterica and uropathogenic Escherichia coli strains, was the main receptor for ferric salmochelin transport.

Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism

SummaryA selection procedure using Mn2+ is described. A high percentage of the Mn2+ resistant mutants had constitutive iron transport systems. By P1 transduction, and complementation with the cloned fur gene it could be shown that nearly all the mutants constitutive in the expression of the operon fusion fiu::λplacMu were only defective in fur. High concentrations of manganese inhibited the derepression of an iron-regulated lac operon fusion. In another iron-regulated lac operon fusion that was inducible by iron, manganese also induced the production of β-galactosidase. Most of the fur mutants isolated (80%) were not able to grow on succinate, fumarate or acetate. After transformation with a fur+ plasmid all 39 mutants tested were able to grow on succinate. In fur mutants the presence of succinate in the growth medium reduced succinate uptake rates by 50%–70%. Succinate dehydrogenase activity was reduced to 10% of that of the parent strain.

Transport of haemin across the cytoplasmic membrane through a haemin-specific periplasmic binding-protein-dependent transport system in Yersinia enterocolitica

The Yersinia enterocolitica O:8 periplasmic binding‐protein‐dependent transport (PBT) system for haemin was cloned and characterized. It consisted of four proteins: the periplasmic haemin‐binding protein HemT, the haemin permease protein HemU, the ATP‐binding hydrophilic protein HemV and the putative haemin‐degrading protein HemS. Y. enterocolitica strains mutated in hemU or hemV genes were unable to use haemin as an iron source whereas those mutated in the hemT gene were able to use haemin as an iron source. As Escherichia coli strains expressing only the haemin outer membrane receptor protein HemR from Y. enterocolitica were capable of using haemin as an iron source the existence of an E. coli K‐12 haemin‐specific PBT system is postulated. The first gene in the Y. enterocolitica haemin‐specific PBT system encoded a protein, HemS, which is probably involved in the degradation of haemin in the cytoplasm. The presence of the hemS gene was necessary to prevent haemin toxicity in E. coli strains that accumulate large amounts of haemin in the cytoplasm. We propose a model of haemin utilization in Y. enterocolitica in which HemT, HemU and HemV proteins transport haemin into the cytoplasm where it is degraded by HemS thereby liberating the iron.

The Zinc-responsive Regulator Zur and Its Control of theznu Gene Cluster Encoding the ZnuABC Zinc Uptake System in Escherichia coli

The synthesis of the Escherichia colizinc transporter, encoded by the znuACB gene cluster, is regulated in response to the intracellular zinc concentration by thezur gene product. Inactivation of the zur gene demonstrated that Zur acts as a repressor when binding Zn2+. Eight chromosomal mutant zur alleles were sequenced to correlate the loss of Zur function with individual mutations. Wild-type Zur and ZurΔ46–91 formed homo- and heterodimers. Dimerization was independent of metal ions since it also occurred in the presence of metal chelators. Using an in vivo titration assay, the znu operator was narrowed down to a 31-base pair region overlapping the translational start site of znuA. This location was confirmed by footprinting assays. Zur directly binds to a single region comprising a nearly perfect palindrome. Zinc chelators completely inhibited and Zn2+ in low concentrations enhanced DNA binding of Zur. No evidence for autoregulation of Zur was found. Zur binds at least 2 zinc ions/dimer specifically. Although most of the mutant Zur proteins bound to the znu operator in vitro, no protection was observed in in vivo footprinting experiments. Analysis of the mutant Zur proteins suggested an amino-terminal DNA contact domain around residue 65 and a dimerization and Zn2+-binding domain toward the carboxyl-terminal end.

Cloning of the repressor protein gene of iron-regulated systems in Escherichia coli K12

SummaryIn Escherichia coli the iron uptake systems are regulated by the fur gene product. The synthesis of the outer membrane proteins fiu, fepA, fecA, fhuA, fhuE and cir is derepressed at low iron concentrations in the medium or constitutive in a fur mutant. The fur gene region cloned into pACYC184 was analysed by restriction analysis, Tn1000 mutagenesis and complementation studies. The presence of fur+ plasmids repressed synthesis of the proteins fepA, fecA, fhuE and cir in a chromosomal fur mutant. More quantitatively, the repression to wild-type levels was shown with lac fusions to the genes fiu, fepA and cir. In minicells an 18,000 dalton protein was identified as the fur gene product. Correlated with the fur protein a slightly smaller protein, possibly a degradation product, was observed. The gene fur was mapped on the E. coli chromosome near nagA at about 15.5 min.

Recent insights into iron import by bacteria

Bacteria are confronted with a low availability of iron owing to its insolubility in the Fe3+ form or its being bound to host proteins. The bacteria cope with the iron deficiency by using host heme or siderophores synthesized by themselves or other microbes. In contrast to most other nutrients, iron compounds are tightly bound to proteins at the cell surfaces, from which they are further translocated by highly specific proteins across the cell wall of gram-positive bacteria and the outer membrane of gram-negative bacteria. Once heme and iron siderophores arrive at the cytoplasmic membrane, they are taken up across the cytoplasmic membrane by ABC transporters. Here we present an outline of bacterial heme and iron siderophore transport exemplified by a few selected cases in which recent progress in the understanding of the transport mechanisms has been achieved.

Ton-dependent colicins and microcins: modular design and evolution

Ton-dependent colicins and microcins are actively taken up into sensitive cells at the expense of energy which is provided by the proton motive force of the cytoplasmic membrane. The Ton system consisting of the proteins TonB, ExbB and ExbD is required for colicin and microcin import. Colicins as well as the outer membrane transport proteins contain proximal to the N-terminus a short sequence, called TonB box, which interacts with TonB and in which point mutants impair uptake. No TonB box is found in microcins. Colicins are composed of functional modules which during evolution have been interchanged resulting in new colicins. The modules define sites of interaction with the outer membrane transport genes, TonB, the immunity proteins, and the activity regions. Six TonB-dependent microcins with different primary structures are processed and exported by highly homologous proteins. Three of these microcins are modified in an unknown way and they have in common specificity for catecholate siderophore receptors.

Dual Repression by Fe2+-Fur and Mn2+-MntR of the mntH Gene, Encoding an NRAMP-Like Mn2+ Transporter in Escherichia coli

The uptake of Mn(2+), a cofactor for several enzymes in Escherichia coli, is mediated by MntH, a proton-dependent metal transporter, which also recognizes Fe(2+) with lower affinity. MntH belongs to the NRAMP family of eukaryotic Fe(2+) and Mn(2+) transporters. In E. coli strains with chromosomal mntH-lacZ fusions, mntH was partially repressed by both Mn(2+) and Fe(2+). Inactivation of fur resulted in the loss of Fe(2+)-dependent repression of mntH transcription, demonstrating that Fe(2+) repression depends on the global iron regulator Fur. However, these fur mutants still showed Mn(2+)-dependent repression of mntH. The Mn(2+)-responsive transcriptional regulator of mntH was identified as the gene product of o155 (renamed MntR). mntR mutants were impaired in Mn(2+) but not Fe(2+) repression of mntH transcription. Binding of purified MntR to the mntH operator was manganese dependent. The binding region was localized by DNase I footprinting analysis and covers a nearly perfect palindrome. The Fur binding site, localized within 22 nucleotides of the mntH operator by in vivo operator titration assays, resembles the Fur-box consensus sequence.